Project Summary Many aspects of human physiology and disease are closely tied to the state of the gut microbiome, yet determining mechanisms by which the microbiome exerts effects at the cellular level is still an open question. Microbes break down dietary fiber, which results in the generation of short chain fatty acids (SCFAs) as metabolic byproducts. These fermentation reactions create an environment in the gastrointestinal tract with high concentrations of SCFAs, which can then act on neighboring host cells. Recently, SCFAs have been detected as chemical modifications on histone proteins, called histone acyl marks, suggesting that a portion of these metabolites enter the nucleus for deposition onto chromatin. While certain histone acyl marks have been reported to positively regulate transcription, including the well-studied histone acetylation, the mechanistic functional role of newer acyl marks such as histone butyrylation are largely unknown. In addition, how SCFAs are regulated in the cell to enter the nucleus and act on chromatin is undetermined. In the mouse intestine, particular acyl modifications on histones are positively correlated with the presence of microbes. In addition, antibiotic treatment reduces levels of histone acyl marks, suggesting select histone acyl marks are dependent on the microbiota. This dependency of histone acyl marks on the microbiota occurs in a tissue-specific manner, with the highest differences observed in the cecum, where the small and large intestines intersect. Now we aim to determine the function of specific microbiota-dependent chromatin acyl marks on gene expression using the mouse gut as a model system. We hypothesize that SCFAs are written onto chromatin as histone acylations in a regulated manner, which mediate key transcriptional programs in response to the microbial environment. To address this hypothesis, the mechanistic function of select histone acyl marks in gene regulation will be investigated, through the use of the mouse gut as a model to study these histone marks. In addition, microbiota-dependent chromatin acyl marks and metabolites will be defined in a comprehensive manner. Lastly, the regulation of histone acyl marks by metabolic enzymes will also be determined. Together, completion of these aims will expand our understanding of the physiological roles of novel histone marks, and will also provide mechanistic insight into how the microbiome impacts the chromatin landscape. Furthermore, this proposal will elucidate the functions of histone acyl marks through chromatin reader protein recruitment and downstream regulation of transcription.